In vitro studies in our laboratory and others have demonstrated direct interactions between Borrelia burgdorferi, the Lyme disease spirochete, and human B and T cells. However, little is known about this interaction in vivo. To assess whether spirochetes interact directly with lymphocytes in mammals, we have developed a mouse model for lymphotrophopism. By repeatedly coincubating spirochetes with primary mouse lymphocytes that were immobilized by adherence to immunomagnetic beads, we were able to preferentially enrich cultures for and against bacteria with constitutive affinity for murine B and T cells. Populations of lymphotropically enriched, stock infectious, and lymphotropically depleted spirochetes were injected intradermally into mice. Lymphocytes were then purified from the blood and spleens of challenged mice and placed into spirochetal culture medium. Cultures of Borrelia burgdorferi were obtained from primary lymphocyte preparations from mice challenged with each of the three populations of spirochetes. Recovery of lymphocyte-associated bacteria occurred within one hour of challenge with enriched bacteria. Lymphocyte preparations from mice challenged with stock infectious and lymphotropiocally depleted bacteria produced positive cultures after one day postchallenge. All lymphocyte preparations were culture negative after one week. These results demonstrate that lymphotropic Borrelia burgdorferi is highly infectious in mice and suggest that associations between spirochetes and lymphocytes occur in vivo. These results also suggest that factors involved in lymphocytic binding may be inducible in vivo. Thus, this system provides an important model for studying the role of such interactions in mammalian infections.